Antisense inhibition of TNFR1 expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of TNFR1. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding TNFR1. Methods of using these compounds for modulation of TNFR1 expression and for treatment of diseases associated with expression of TNFR1 are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods of modulatingthe expression of TNFR1. In particular, this invention relates toantisense compounds, particularly oligonucleotides, specificallyhybridizable with nucleic acids encoding human TNFR1. Sucholigonucleotides have been shown to modulate the expression of TNFR1.

BACKGROUND OF THE INVENTION

One of the principal mechanisms by which cellular regulation is effectedis through the transduction of extracellular signals into intracellularsignals that in turn modulate biochemical pathways. Examples of suchextracellular signaling molecules include growth factors, cytokines, andchemokines. The cell surface receptors of these molecules and theirassociated signal transduction pathways are therefore one of theprincipal means by which cellular behavior is regulated. Becausecellular phenotypes are largely influenced by the activity of thesepathways, it is currently believed that a number of disease statesand/or disorders are a result of either aberrant activation orfunctional mutations in the molecular components of signal transductionpathways.

For example, the polypeptide cytokine tumor necrosis factor (TNF) isnormally produced during infection, injury, or invasion where it servesas a pivotal mediator of the inflammatory response. In recent years, anumber of in vivo animal and human studies have demonstrated thatoverexpression of TNF by the host in response to disease and infectionis itself responsible for the pathological consequences associated withthe underlying disease. For example, septic shock as a result of massivebacterial infection has been attributed to infection-induced expressionof TNF. Thus, systemic exposure to TNF at levels comparable to thosefollowing massive bacterial infection has been shown to result in aspectrum of symptoms (shock, tissue injury, capillary leakage, hypoxia,pulmonary edema, multiple organ failure and high mortality rate) that isvirtually indistinguishable from septic shock syndrome (Tracey andCerami, Annu. Rev. Med., 1994, 45, 491-503). Further evidence has beenprovided in animal models of septic shock, in which it has beendemonstrated that systemic exposure to anti-TNF neutralizing antibodiesblocks bacterial-induced sepsis (Tracey and Cerami, Annu. Rev. Med.,1994, 45, 491-503). In addition to these acute effects, chronic exposureto low doses of TNF results in a syndrome of cachexia marked byanorexia, weight loss, dehydration and depletion of whole-body proteinand lipid. Chronic production of TNF has been implicated in a number ofdiseases including AIDS and cancer (Tracey and Cerami, Annu. Rev. Med.,1994, 45, 491-503). To date, two distinct TNF cell surface receptors,known as TNFR1 and TNFR2, have been described. Molecular analysis ofTNFR1 and TNFR2 have shown that the two receptors share little homologyin their intracellular domains and appear to activate distinctintracellular pathways (Tracey and Cerami, Annu. Rev. Med., 1994, 45,491-503).

Recent studies with transgenic TNFR1 knockout mice have demonstratedthat signaling through TNFR1 plays an important role in the clearing oflow-level bacterial infection as well as TNF-induced septic shockfollowing high-level bacterial infection (Lotz et al., J. Leukoc. Biol.,1996, 60, 1-7). These findings indicate that agents which can inhibitsignaling through the TNFR1 receptor may serve as useful targetsinhibitors of TNF induced toxicities such as septic shock.

Antisense oligonucleotide inhibition of TNFRl has proven to be a usefultool in understanding the role of TNFR1 stimulation in cytokineinduction and cell proliferation. Ojwang et. al. have disclosed partialphosphorothioate antisense oligodeoxynucleotides containing C-5 propynylor hexynyl derivatives of 2'-deoxyuridine which caused attenuation ofTNFR1 mRNA and protein and inhibited TNF-alpha induced expression ofIL-6 in MRC-5 cells (Ojwang et al., Biochemistry, 1997, 36, 6033-6045).These oligonucleotides were targeted to the poly(A) signal site of TNFR1mRNA. A uniform phosphorothioate oligodeoxynucleotide targeted to thetranslation initiation codon of TNFR1 was found to have no effect.

There remains a long-felt need for improved compositions and methods forinhibiting TNFR1 gene expression.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds, particularlyoligonucleotides, which are targeted to a nucleic acid encoding TNFR1,and which modulate the expression of TNFR1. Pharmaceutical and othercompositions comprising the antisense compounds of the invention arealso provided. Further provided are methods of modulating the expressionof TNFR1 in cells or tissues comprising contacting said cells or tissueswith one or more of the antisense compounds or compositions of theinvention. Further provided are methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of TNFR1 by administering atherapeutically or prophylactically effective amount of one or more ofthe antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric antisense compounds,particularly oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding TNFR1, ultimately modulating the amountof TNFRl produced. This is accomplished by providing antisense compoundswhich specifically hybridize with one or more nucleic acids encodingTNFR1. As used herein, the terms "target nucleic acid" and "nucleic acidencoding TNFR1" encompass DNA encoding TNFR1, RNA (including pre-mRNAand mRNA) transcribed from such DNA, and also cDNA derived from suchRNA. The specific hybridization of an oligomeric compound with itstarget nucleic acid interferes with the normal function of the nucleicacid. This modulation of function of a target nucleic acid by compoundswhich specifically hybridize to it is generally referred to as"antisense." The functions of DNA to be interfered with includereplication and transcription. The functions of RNA to be interferedwith include all vital functions such as, for example, translocation ofthe RNA to the site of protein translation, translation of protein fromthe RNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The overall effect of such interference with target nucleic acidfunction is modulation of the expression of TNFR1. In the context of thepresent invention, "modulation" means either an increase (stimulation)or a decrease (inhibition) in the expression of a gene. In the contextof the present invention, inhibition is the preferred form of modulationof gene expression and mRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense."Targeting" an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding TNFR1. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5'-AUG (in transcribed mRNAmolecules; 5'-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the "AUG codon," the "startcodon" or the "AUG start codon." A minority of genes have a translationinitiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, theterms "translation initiation codon" and "start codon" can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine(prokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, "start codon" and "translationinitiation codon" refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding TNFR1, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or"stop codon") of a gene may have one of three sequences, i.e., 5'-UAA,5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAGand 5'-TGA, respectively). The terms "start codon region" and"translation initiation codon region" refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5' or 3') from a translationinitiation codon. Similarly, the terms "stop codon region" and"translation termination codon region" refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5'or 3') from a translationtermination codon.

The open reading frame (ORF) or "coding region," which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5'untranslatedregion (5'UTR), known in the art to refer to the portion of an mRNA inthe 5' direction from the translation initiation codon, and thusincluding nucleotides between the 5' cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene)and the 3' untranslated region (3' UTR), known in the art to refer tothe portion of an mRNA in the 3' direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3' end of an mRNA or correspondingnucleotides on the gene). The 5' cap of an mRNA comprises anN7-methylated guanosine residue joined to the 5'-most residue of themRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA isconsidered to include the 5' cap structure itself as well as the first50 nucleotides adjacent to the cap. The 5' cap region may also be apreferred target region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as "introns," which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as "exons" and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, "hybridization" means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. "Complementary," as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, "specifically hybridizable" and "complementary" are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotides have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans. In the context of this invention, the term"oligonucleotide" refers to an oligomer or polymer of ribonucleic acid(RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This termincludes oligonucleotides composed of naturally-occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally-occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for nucleic acidtarget and increased stability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 30 nucleobases.Particularly preferred are antisense oligonucleotides comprising fromabout 8 to about 30 nucleobases (i.e. from about 8 to about 30 linkednucleosides). As is known in the art, a nucleoside is a base-sugarcombination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. In turn the respective ends of thislinear polymeric structure can be further joined to form a circularstructure, however, open linear structures are generally preferred.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3'to 5' phosphodiester linkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3'-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3'-5' linkages, 2'-5' linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3'-5' to 5'-3'or 2'-5' to 5'-2'. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; and 5,697,248, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.:5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular --CH₂ --NH--O--CH₂ --, --CH₂--N(CH₃)--O--CH₂ -- [known as a methylene (methylimino) or MMIbackbone], --CH₂ --O--N(CH₃) --CH₂ --,--CH₂ --N(CH₃)--N (CH₃) --CH₂ --and --O--N(CH₃) --CH₂ --CH₂ -- [wherein the native phosphodiesterbackbone is represented as --O--P--O--CH₂ --] of the above referencedU.S. Pat. No. 5,489,677, and the amide backbones of the above referencedU.S. Pat. No. 5,602,240. Also preferred are oligonucleotides havingmorpholino backbone structures of the above-referenced U.S. Pat. No.5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2' position: OH; F; O-, S-, or N-alkyl, O-, S-, or N-alkenyl, O, S-or N-alkynyl, or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n) O]_(m) CH₃,O(CH₂)_(n) OCH₃, O(CH₂)_(n) NH₂, O (CH₂)_(n) CH₃,O(CH₂)_(n) ONH₂, andO(CH₂)_(n) ON[(CH₂)_(n) CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2'position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂ CH₃, ONO₂,NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2'-methoxyethoxy (2'--O--CH₂ CH₂ OCH₃, also known as2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. A further preferredmodification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2'-DMAOE, as described in U.S. patentapplication Ser. No. 09/016,520, filed on Jan. 30, 1998, which iscommonly owned with the instant application and the contents of whichare herein incorporated by reference.

Other preferred modifications include 2'-methoxy (2'-O-CH₃),2'-aminopropoxy (2'-OCH₂ CH₂ CH₂ NH₂) and 2'-fluoro (2'-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3' position of the sugar on the 3'terminal nucleotide or in 2'-5' linked oligonucleotides and the 5'position of 5' terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugars structures include, but are not limited to, U.S.Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference, and allowed U.S. patent application Ser. No. 08/468,037,filed on Jun. 5, 1995, U.S. Pat. No. X,XXX,XXX, which is commonly ownedwith the instant application and is also herein incorporated byreference.

Oligonucleotides may also include nucleobase (often referred to in theart simply as "base") modifications or substitutions. As used herein,"unmodified" or "natural" nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J.I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2'-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference, and allowed U.S. patent application Ser. No.08/762,488, filed on Dec. 10, 1996, U.S. Pat. No. X,XXX,XXX, which iscommonly owned with the instant application and also herein incorporatedby reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. "Chimeric"antisense compounds or "chimeras," in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference, and allowed U.S.patent application Ser. No. 08/465,880, filed on Jun. 6, 1995, U.S. Pat.No. X,XXX,XXX, which is commonly owned with the instant application andalso herein incorporated by reference.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. No.: 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term "prodrug" indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 to Imbach et al.

The term "pharmaceutically acceptable salts" refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., "Pharmaceutical Salts," J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a "pharmaceutical addition salt"includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine. The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of TNFR1 is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingTNFR1, enabling sandwich and other assays to easily be constructed toexploit this fact. Hybridization of the antisense oligonucleotides ofthe invention with a nucleic acid encoding TNFR1 can be detected bymeans known in the art. Such means may include conjugation of an enzymeto the oligonucleotide, radiolabelling of the oligonucleotide or anyother suitable detection means. Kits using such detection means fordetecting the level of TNFR1 in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 21-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions and/or formulations comprising theoligonucleotides of the present invention may also include penetrationenhancers in order to enhance the alimentary delivery of theoligonucleotides. Penetration enhancers may be classified as belongingto one of five broad categories, i.e., fatty acids, bile salts,chelating agents, surfactants and non-surfactants (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, 8, 91-192; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33).One or more penetration enhancers from one or more of these broadcategories may be included. Penetration enhancers are described inpending U.S. patent application Ser. No. 08/886,829, filed on Jul. 1,1997, U.S. Pat. No. X,XXX,XXX, and pending U.S. patent application Ser.No. 08/961,469, filed on Oct. 31, 1997, U.S. Pat. No. X,XXX,XXX, both ofwhich are commonly owned with the instant application and both of whichare herein incorporated by reference.

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, 8:2, 91-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1, 1-33; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651-654). Examples of some presentlypreferred fatty acids are sodium caprate and sodium laurate, used singlyor in combination at concentrations of 0.5 to 5%.

Preferred penetration enhancers are disclosed in pending U.S. patentapplication Ser. No. 08/886,829, filed on Jul. 1, 1997, U.S. Pat. No.X,XXX,XXX, which is commonly owned with the instant application andwhich is herein incorporated by reference.

The physiological roles of bile include the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al., eds., McGraw-Hill, New York, NY, 1996, pages934-935). Various natural bile salts, and their synthetic derivatives,act as penetration enhancers. Thus, the term "bile salt" includes any ofthe naturally occurring components of bile as well as any of theirsynthetic derivatives. Preferred bile salts are described in pendingU.S. patent application Ser. No. 08/886,829, filed on Jul. 1, 1997, U.S.Pat. No. X,XXX,XXX, which is commonly owned with the instant applicationand which is herein incorporated by reference. A presently preferredbile salt is chenodeoxycholic acid (CDCA) (Sigma Chemical Company, St.Louis, Mo.), generally used at concentrations of 0.5 to 2%.

Complex formulations comprising one or more penetration enhancers may beused. For example, bile salts may be used in combination with fattyacides to make complex formulations. Preferred combinations include CDCAcombined with sodium caprate or sodium laurate (generally 0.5 to 5%).

Chelating agents include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, 8:2, 92-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1, 1-33; Buur et al., J.Control Rel., 1990, 14, 43-51). Chelating agents have the addedadvantage of also serving as DNase inhibitors.

Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8:2,92-191); and perfluorochemical emulsions, such as FC-43 (Takahashi etal., J. Pharm. Phamacol., 1988, 40, 252-257).

Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl-and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, 8:2, 92-191); andnon-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

As used herein, "carrier compound" refers to a nucleic acid, or analogthereof, which is inert (i.e., does not possess biological activity perse) but is recognized as a nucleic acid by in vivo processes that reducethe bioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioated oligonucleotide in hepatic tissue is reducedwhen it is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4¹-isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., AntisenseRes. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. AcidDrug Dev., 1996, 6, 177-183).

In contrast to a carrier compound, a "pharmaceutically acceptablecarrier" (excipient) is a pharmaceutically acceptable solvent,suspending agent or any other pharmacologically inert vehicle fordelivering one or more nucleic acids to an animal. The pharmaceuticallyacceptable carrier may be liquid or solid and is selected with theplanned manner of administration in mind so as to provide for thedesired bulk, consistency, etc., when combined with a nucleic acid andthe other components of a given pharmaceutical composition. Typicalpharmaceutically acceptable carriers include, but are not limited to,binding agents (e.g., pregelatinized maize starch, polyvinyl-pyrrolidoneor hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose andother sugars, microcrystalline cellulose, pectin, gelatin, calciumsulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate,etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidalsilicon dioxide, stearic acid, metallic stearates, hydrogenatedvegetable oils, corn starch, polyethylene glycols, sodium benzoate,sodium acetate, etc.); disintegrates (e.g., starch, sodium starchglycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate,etc.). Sustained release oral delivery systems and/or enteric coatingsfor orally administered dosage forms are described in U.S. Pat. Nos.4,704,295; 4,556,552; 4,309,406; and 4,309,404.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional compatible pharmaceutically-activematerials such as, e.g., antipruritics, astringents, local anestheticsor anti-inflammatory agents, or may contain additional materials usefulin physically formulating various dosage forms of the composition ofpresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of theinvention.

Regardless of the method by which the antisense compounds of theinvention are introduced into a patient, colloidal dispersion systemsmay be used as delivery vehicles to enhance the in vivo stability of thecompounds and/or to target the compounds to a particular organ, tissueor cell type. Colloidal dispersion systems include, but are not limitedto, macromolecule complexes, nanocapsules, microspheres, beads andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, liposomes and lipid:oligonucleotide complexes ofuncharacterized structure. A preferred colloidal dispersion system is aplurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layer(s) made up of lipidsarranged in a bilayer configuration (see, generally, Chonn et al.,Current Op. Biotech., 1995, 6, 698-708). Liposome preparation isdescribed in pending U.S. patent application Ser. No. 08/961,469, filedon Oct. 31, 1997, U.S. Pat. No. X,XXX,XXX, which is commonly owned withthe instant application and which is herein incorporated by reference.

Certain embodiments of the invention provide for liposomes and othercompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include, but are notlimited to, anticancer drugs such as daunorubicin, dactinomycin,doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil,melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine(CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228). Antiinflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. See, generally, The Merck Manual of Diagnosis and Therapy,15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and46-49, respectively). Other non-antisense chemotherapeutic agents arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Examples of antisenseoligonucleotides include, but are not limited to, those directed to thefollowing targets as disclosed in the indicated U.S. Patents, or pendingU.S. applications, which are commonly owned with the instant applicationand are hereby incorporated by reference, or the indicated published PCTapplications: raf (WO 96/39415, WO 95/32987 and U.S. Pat. Nos.5,563,255, issued Oct. 8, 1996, and U.S. Pat. No. 5,656,612, issued Aug.12, 1997), the p120 nucleolar antigen (WO 93/17125 and U.S. Pat. No.5,656,743, issued Aug. 12, 1997), protein kinase C (WO 95/02069, WO95/03833 and WO 93/19203), multidrug resistance-associated protein (WO95/10938 and U.S. Pat. No. 5,510,239, issued Mar. 23, 1996), subunits oftranscription factor AP-1 (pending application U.S. Ser. No. 08/837,201,filed Apr. 14, 1997), Jun kinases (pending application U.S. Ser. No.08/910,629, filed Aug. 13, 1997), MDR-1 (multidrug resistanceglycoprotein; pending application U.S. Ser. No. 08/731,199, filed Sep.30, 1997), HIV (U.S. Pat. Nos. 5,166,195, issued Nov. 24, 1992 and5,591,600, issued Jan. 7, 1997), herpesvirus (U.S. Pat. No. 5,248,670,issued Sep. 28, 1993 and U.S. Pat. No. 5,514,577, issued May 7, 1996),cytomegalovirus (U.S. Pat. No. 5,442,049, issued Aug. 15, 1995 and5,591,720, issued Jan. 7, 1997), papillomavirus (U.S. Pat. 5,457,189,issued Oct. 10, 1995), intercellular adhesion molecule-1 (ICAM-1) (U.S.Pat. No. 5,514,788, issued May 7, 1996), 5-lipoxygenase (U.S. Pat. No.5,530,114, issued Jun. 25, 1996) and influenzavirus (U.S. Pat. No.5,580,767, issued Dec. 3, 1996). Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀ s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES

Example 1

Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and2'-alkoxy amidites

21-Deoxy and 21-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham Mass. or GlenResearch, Inc. Sterling Va.). Other 2¹ -O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. No. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2'-alkoxy amidites, the standard cycle for unmodified oligonucleotideswas utilized, except the wait step after pulse delivery of tetrazole andbase was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2'-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2'-Fluoro amidites

2'-Fluorodeoxyadenosine amidites

2'-fluoro oligonucleotides were synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2'-deoxy-2'-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2'-alpha-fluoro atom is introduced by a S_(N) 2-displacement of a2'-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3',5'-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5'-dimethoxytrityl- (DMT) and5'-DMT-3'-phosphoramidite intermediates.

2'-Fluorodeoxyguanosine

The synthesis of 2'-deoxy-2'-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5'-DMT- and5'-DMT-3'-phosphoramidites.

2'-Fluorouridine

Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by themodification of a literature procedure in which2,2'-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5'-DMT and 5'-DMT-3'phosphoramidites.

2'-Fluorodeoxycytidine

2'-deoxy-21-fluorocytidine was synthesized via amination of2'-deoxy-2'-fluorouridine, followed by selective protection to giveN4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were used toobtain the 5'-DMT and 5'-DMT-3'phosphoramidites.

2'-O-(2-Methoxyethyl) modified amidites

2'-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.).

2'-O-Methoxyethyl-5-methyluridine

2,2'-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃ CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂ Cl₂ /acetone/MeOH (20:5:3)containing 0.5% Et₃ NH. The residue was dissolved in CH₂ Cl₂ (250 mL)and adsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃ CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂ SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃ NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃ CN (700 mL) and set aside. Triethylamine (189 mL,1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃ CN (1L), cooled to -5° C. and stirred for 0.5 h using an overhead stirrer.POCl₃ was added dropwise, over a 30 minute period, to the stirredsolution maintained at 0-10° C., and the resulting mixture stirred foran additional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

2'-O-Methoxyethyl-5N-O-dimethoxytrityl-5-methylcytidine

A solution of3'-0-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄ OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tlc showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃ NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amidite

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂ Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂ Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2'-(Aminooxyethyl) nucleoside amidites and 2'-(dimethylaminooxyethyl)nucleoside amidites

Aminooxyethyl and dimethylaminooxyethyl amidites are prepared as per themethods of United States patent applications Ser. No. 10/037,143, filedFeb. 14, 1998, and Ser. No. 09/016,520, filed Jan. 30, 1998, each ofwhich is commonly owned with the instant application and is hereinincorporated by reference.

Example 2

Oligonucleotide synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 hr), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4

PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the "gap" segment oflinked nucleosides is positioned between 5' and 3' "wing" segments oflinked nucleosides and a second "open end" type wherein the "gap"segment is located at either the 3' or the 5' terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas "gapmers" or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as "hemimers" or "wingmers."

[2'-O-Me]--[2'-deoxy]--[2'-O-Me] Chimeric

Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and2'-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA portion and5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3' wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2'-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 Ammonia/Ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2' positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to 1/2 volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometer.

[2'-O-(2-Methoxyethyl)]--[2'-deoxy]--[2'-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2'-O-(2-methoxyethyl)]--[2'-deoxy]--[-2-C-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2'-O-methyl chimeric oligonucleotide, with thesubstitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methylamidites.

[2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxyPhosphorothioate]--[2'-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[21-O-(2-methoxyethyl phosphodiester]--[2'-deoxyphosphorothioate]--[21-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2'-O-methyl chimeric oligonucleotide with the substitution of2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹ P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

Oligonucleotide Synthesis--96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected betacyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄ OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

Oligonucleotide Analysis--96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing Electrospray-MassSpectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

Cell culture and oligonucleotide treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing four cell types are provided for illustrative purposes, butother cell types can be routinely used.

T-24 cells:

The transitional cell bladder carcinoma cell line T-24 was obtained fromthe American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cellswere routinely cultured in complete McCoy's 5A basal media (Gibco/LifeTechnologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units permL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence. Cells were seeded into96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/wellfor use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trysinization anddilution when they reached 90% confluence.

NHDF cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cell were routinelymaintained for up to 10 passages as recommended by the supplier.

Treatment with antisense compounds:

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired oligonucleotide at a final concentration of150 nM. After 4 hours of treatment, the medium was replaced with freshmedium. Cells were harvested 16 hours after oligonucleotide treatment.

Example 10

Antisense inhibition of TNFR1 expression- phosphorothioateoligodeoxynucleotides

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human TNFR1 RNA, usingpublished sequences (GenBank accession number X55313, incorporatedherein as SEQ ID NO: 1). The oligonucleotides are shown in Table 1.Target sites are indicated by nucleotide numbers, as given in thesequence source reference (Genbank accession no. X55313), to which theoligonucleotide binds. All compounds in Table 1 areoligodeoxynucleotides with phosphorothioate backbones (internucleosidelinkages) throughout. The compounds were analyzed for effect on TNFR1mRNA levels by quantitative real-time PCR as described in subsequentexamples. Data are averages from three experiments.

                  TABLE 1                                                         ______________________________________                                        Inhibition of TNFR1 mRNA levels by phosphorothioate                             oligodeoxynucleotides                                                                                            %      SEQ                                   TARGET  Inhibi- ID                                                          ISIS# REGION SITE SEQUENCE tion NO.                                         ______________________________________                                        18875 5' UTR  37      TTCTCTGGACTGAGGCTC                                                                         19     8                                      - 18876 5' UTR 68 TCCCCTCCTCTCTGCTTT 5 9                                      - 18877 5' UTR 109 AGACTCGGGCATAGAGAT 0 10                                    - 18878 5' UTR 114 GGTTGAGACTCGGGCATA 40 11                                   - 18879 5' UTR 118 TGAGGGTTGAGACTCGGG 2 12                                    - 18880 5' UTR 123 ACAGTTGAGGGTTGAGAC 30 13                                   - 18881 5' UTR 127 GGTGACAGTTGAGGGTTG 8 14                                    - 18882 5' UTR 196 GCAGTGTGGCAGCGGCAG 53 15                                   - 18883 5' UTR 199 AGGGCAGTGTGGCAGCGG 53 16                                   - 18884 5' UTR 202 CTCAGGGCAGTGTGGCAG 61 17                                   - 18885 5' UTR 207 TTGGGCTCAGGGCAGTGT 0 18                                    - 18886 5' UTR 210 CATTTGGGCTCAGGGCAG 9 19                                    - 18887 Coding 262 GTCAGGCACGGTGGAGAG 0 20                                    - 18888 Coding 266 GCAGGTCAGGCACGGTGG 16 21                                   - 18889 Coding 272 GCAGCAGCAGGTCAGGCA 37 22                                   - 18890 Coding 276 AGCGGCAGCAGCAGGTCA 0 23                                    - 18891 Coding 280 CACCAGCGGCAGCAGCAG 21 24                                   - 18892 Coding 286 CAGGAGCACCAGCGGCAG 46 25                                   - 18893 Coding 306 TATATTCCCACCAACAGC 25 26                                   - 18894 Coding 356 TCTTCTCCCTGTCCCCTA 13 27                                   - 18895 Coding 403 ATTATTTTGAGGGTGGAT 0 28                                    - 18896 Coding 435 GTTCCTTTGTGGCACTTG 12 29                                   - 18897 Coding 440 AGTAGGTTCCTTTGTGGC 46 30                                   - 18898 Coding 460 GCCTGGACAGTCATTGTA 0 31                                    - 18899 Coding 480 CAGTCCGTATCCTGCCCC 26 32                                   - 18900 Coding 500 AGCCGCTCTCACACTCCC 36 33                                   - 18901 Coding 516 TCTGAAGCGGTGAAGGAG 0 34                                    - 18902 Coding 521 GGTTTTCTGAAGCGGTGA 17 35                                   - 18903 Coding 525 AGGTGGTTTTCTGAAGCG 0 36                                    - 18904 Coding 530 GTCTGAGGTGGTTTTCTG 34 37                                   - 18905 Coding 537 AGGCAGTGTCTGAGGTGG 0 38                                    - 18906 Coding 542 AGCTGAGGCAGTGTCTGA 27 39                                   - 18907 Coding 565 CATTTCCTTTCGGCATTT 13 40                                   - 18908 Coding 569 GACCCATTTCCTTTCGGC 26 41                                   - 18909 Coding 574 CACCTGACCCATTTCCTT 46 42                                   - 18910 Coding 635 GGTACTGGTTCTTCCTGC 14 43                                   - 18911 Coding 654 TTTTCACTCCAATAATGC 0 44                                    - 18912 Coding 693 CCATTGAGGCAGAGGCTG 48 45                                   - 18913 Coding 699 ACGGTCCCATTGAGGCAG 34 46                                   - 18914 Coding 732 ACGGTGTTCTGTTTCTCC 7 47                                    - 18915 Coding 786 CTACAGGAGACACACTCG 28 48                                   - 18916 Coding 796 CTTACAGTTACTACAGGA 21 49                                   - 18917 Coding 802 GCTTTTCTTACAGTTACT 10 50                                   - 18918 Coding 807 TCCAGGCTTTTCTTACAG 0 51                                    - 18919 Coding 845 TAACATTCTCAATCTGGG 0 52                                    - 18920 Coding 873 ACTGTGGTGCCTGAGTCC 31 53                                   - 18921 Coding 906 CAAAGACCAAAGAAAATG 29 54                                   - 18922 Coding 911 AAAGGCAAAGACCAAAGA 31 55                                   - 18923 Coding 921 AGGAGGGATAAAAGGCAA 22 56                                   - 18924 Coding 929 CAATGAAGAGGAGGGATA 21 57                                   - 18925 Coding 935 TTAAACCAATGAAGAGGA 28 58                                   - 18926 Coding 952 CCGTTGGTAGCGATACAT 30 59                                   - 18927 Coding 992 TCGATTTCCCACAAACAA 1 60                                    - 18928 Coding 1033 CTTAGTAGTAGTTCCTTC 15 61                                  - 18929 Coding 1075 GAAGCCTGGAGTGGGACT 48 62                                  - 18930 Coding 1098 GGACTGAAGCCCAGGGTG 12 63                                  - 18931 Coding 1113 GTGGAACTGGGCACGGGA 4 64                                   - 18932 Coding 1118 TGAAGGTGGAACTGGGCA 27 65                                  - 18933 Coding 1127 AGCTGGAGGTGAAGGTGG 0 66                                   - 18934 Coding 1162 CGCAAAGTTGGGACAGTC 30 67                                  - 18935 Coding 1184 GTGCCACCTCTCTGCGGG 0 68                                   - 18936 Coding 1269 CTGTCCTCCCACTTCTGA 16 69                                  - 18937 Coding 1290 AGGCTCTGTGGCTTGTGG 47 70                                  - 18938 Coding 1389 TCGTGGTCGCTCAGCCCT 28 71                                  - 18939 Coding 1465 CCGCCTCCAGGTCGCCAG 0 72                                   - 18940 Coding 1537 GCAGCCCAGCAGGTCCAT 32 73                                  - 18941 Coding 1545 TCCTCCAGGCAGCCCAGC 41 74                                  - 18942 Coding 1604 ATCTGAGAAGACTGGGCG 0 75                                   - 18943 Coding 1707 GCTCCTGCTTGCCCCTGC 43 76                                  - 18944 Coding 1732 GTTAGCACCAAGTAGGCG 11 77                                  - 18945 Coding 1842 CGCAAACCACCCACTCAG 51 78                                  - 18946 Coding 1847 ATCCTCGCAAACCACCCA 29 79                                  - 18947 Coding 1859 ATAGCGTCCCTCATCCTC 34 80                                  - 18948 Coding 1925 CTCAGGGACGAACCAGGG 3 81                                   - 18949 Coding 1930 AAAGGCTCAGGGACGAAC 42 82                                  - 18950 Coding 1979 ACAAAACAAAACAAAACA 27 83                                  - 18951 Coding 2016 GCCAAGTTTCTATTAGTG 10 84                                  - 18952 Coding 2033 GCAGAGGGCACAGGAGTG 24 85                                  - 18953 Coding 2039 GTCCAGGCAGAGGGCACA 21 86                                  - 18954 Coding 2043 GCTTGTCCAGGCAGAGGG 18 87                                  - 18955 Coding 2071 TGCCTTAGGACAGTTCAG 20 88                                  - 18956 Coding 2085 TCCGTGCTCGCCCCTGCC 19 89                                  - 18957 Coding 2089 TTGTTCCGTGCTCGCCCC 41 90                                  - 18958 Coding 2097 AGGCCCCATTGTTCCGTG 0 91                                ______________________________________                                    

As shown in Table 1, SEQ ID NOs 11, 15, 16, 17, 22, 25, 30, 33, 42, 45,62, 70, 74, 76, 78, 82 and 90 demonstrated at least 35% inhibition ofTNFR1 expression in this assay and are therefore preferred.

Example 11

Analysis of oligonucleotide inhibition of TNFR1 expression

Antisense modulation of TNFR1 expression can be assayed in a variety ofways known in the art. For example, TNFR1 mRNA levels can be quantitatedby, e.g., Northern blot analysis, competitive polymerase chain reaction(PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+mRNA. Methods of RNA isolation are taught in, for example,Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northernblot analysis is routine in the art and is taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative(PCR) can be conveniently accomplished using the commercially availableABI PRISM™ 7700 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions. Other methods of PCR are also known in the art.

TNFR1 protein levels can be quantitated in a variety of ways well knownin the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).Antibodies directed to TNFR1 can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 12

Poly(A)+mRNA isolation

Poly(A)+mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 13

Total RNA Isolation

Total mRNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pippeting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY ₉₆ ™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

Example 14

Real-time Quantitative PCR Analysis of TNFR1 mRNA Levels

Quantitation of TNFR1 mRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE or FAM, PE-Applied Biosystems, Foster City, Calif.) isattached to the 5'end of the probe and a quencher dye (e.g., TAMRA,PE-Applied Biosystems, Foster City, Calif.) is attached to the 3' end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3' quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5'-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular (six-second) intervals bylaser optics built into the ABI PRISM™ 7700 Sequence Detection System.In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1×TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLpoly(A) mRNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension).

For TNFR1 the PCR primers were:

forward primer: GCTTCAGAAAACCACCTCAGACA (SEQ ID No. 2)

reverse primer: CCGGTCCACTGTGCAAGAA (SEQ ID No. 3) and the PCR probewas: FAM-TCAGCTGCTCCAAATGCCGAAAGG-TAMRA (SEQ ID No. 4) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye.

For GAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID No. 5)

reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID No. 6)and the PCR probewas: 5' JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3' (SEQ ID No. 7) where JOE(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye.

Example 15

Northern blot analysis of TNFR1 mRNA levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST BII Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.).

Membranes were probed using QUICKHYB™ hybridization solution(Stratagene, La Jolla, Calif.) using manufacturer's recommendations forstringent conditions with a TNFR1 specific probe prepared by PCR usingthe forward primer GCTTCAGAAAACCACCTCAGACA (SEQ ID No. 2) and thereverse primer CCGGTCCACTGTGCAAGAA (SEQ ID No. 3). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) RNA(Clontech, Palo Alto, Calif.). Hybridized membranes were visualized andquantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3(Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to G3PDHlevels in untreated controls.

Example 16

Western blot analysis of TNFR1 protein levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 hr after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to TNFR1 is used, with aradiolabelled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 17

Antisense inhibition of TNFR1 expression- phosphorothioate 2'-MOE gapmeroligonucleotides

In accordance with the present invention, a second series ofoligonucleotides targeted to human TNFR1 were synthesized. Theoligonucleotide sequences are shown in Table 2. Target sites areindicated by nucleotide numbers, as given in the sequence sourcereference (Genbank accession no. X55313), to which the oligonucleotidebinds.

All compounds in Table 2 are chimeric oligonucleotides ("gapmers") 18nucleotides in length, composed of a central "gap" region consisting often 21-deoxynucleotides, which is flanked on both sides (5' and 3'directions) by four-nucleotide "wings." The wings are composed of2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide.Cytidine residues in the 2'-MOE wings are 5-methylcytidines.

Data were obtained by real-time quantitative PCR as described inprevious examples and are averaged from three experiments.

                  TABLE 2                                                         ______________________________________                                        Inhibition of TNFR1 mRNA levels by chimeric phospho-                            rothioate oligonucleotides having                                             2'-MOE wings and a deoxy gap                                                                                     %      SEQ                                   TARGET  Inhibi- ID                                                          ISIS# REGION SITE SEQUENCE tion NO.                                         ______________________________________                                        19463 5' UTR  37      TTCTCTGGACTGAGGCTC                                                                         72     8                                      - 19464 5' UTR 68 TCCCCTCCTCTCTGCTTT 9 9                                      - 19465 5' UTR 109 AGACTCGGGCATAGAGAT 18 10                                   - 19466 5' UTR 114 GGTTGAGACTCGGGCATA 95 11                                   - 19467 5' UTR 118 TGAGGGTTGAGACTCGGG 28 12                                   - 19468 5' UTR 123 ACAGTTGAGGGTTGAGAC 66 13                                   - 19469 5' UTR 127 GGTGACAGTTGAGGGTTG 42 14                                   - 19470 5' UTR 196 GCAGTGTGGCAGCGGCAG 78 15                                   - 19471 5' UTR 199 AGGGCAGTGTGGCAGCGG 76 16                                   - 19472 5' UTR 202 CTCAGGGCAGTGTGGCAG 90 17                                   - 19473 5' UTR 207 TTGGGCTCAGGGCAGTGT 48 18                                   - 19474 5' UTR 210 CATTTGGGCTCAGGGCAG 70 19                                   - 19475 Coding 262 GTCAGGCACGGTGGAGAG 66 20                                   - 19476 Coding 266 GCAGGTCAGGCACGGTGG 91 21                                   - 19477 Coding 272 GCAGCAGCAGGTCAGGCA 85 22                                   - 19478 Coding 276 AGCGGCAGCAGCAGGTCA 93 23                                   - 19479 Coding 280 CACCAGCGGCAGCAGCAG 65 24                                   - 19480 Coding 286 CAGGAGCACCAGCGGCAG 60 25                                   - 19481 Coding 306 TATATTCCCACCAACAGC 58 26                                   - 19482 Coding 356 TCTTCTCCCTGTCCCCTA 42 27                                   - 19483 Coding 403 ATTATTTTGAGGGTGGAT 75 28                                   - 19484 Coding 435 GTTCCTTTGTGGCACTTG 88 29                                   - 19485 Coding 440 AGTAGGTTCCTTTGTGGC 78 30                                   - 19486 Coding 460 GCCTGGACAGTCATTGTA 80 31                                   - 19487 Coding 480 CAGTCCGTATCCTGCCCC 66 32                                   - 19488 Coding 500 AGCCGCTCTCACACTCCC 86 33                                   - 19489 Coding 516 TCTGAAGCGGTGAAGGAG 52 34                                   - 19490 Coding 521 GGTTTTCTGAAGCGGTGA 92 35                                   - 19491 Coding 525 AGGTGGTTTTCTGAAGCG 82 36                                   - 19492 Coding 530 GTCTGAGGTGGTTTTCTG 91 37                                   - 19493 Coding 537 AGGCAGTGTCTGAGGTGG 96 38                                   - 19494 Coding 542 AGCTGAGGCAGTGTCTGA 79 39                                   - 19495 Coding 565 CATTTCCTTTCGGCATTT 41 40                                   - 19496 Coding 569 GACCCATTTCCTTTCGGC 93 41                                   - 19497 Coding 574 CACCTGACCCATTTCCTT 63 42                                   - 19498 Coding 635 GGTACTGGTTCTTCCTGC 79 43                                   - 19499 Coding 654 TTTTCACTCCAATAATGC 9 44                                    - 19500 Coding 693 CCATTGAGGCAGAGGCTG 0 45                                    - 19501 Coding 699 ACGGTCCCATTGAGGCAG 81 46                                   - 19502 Coding 732 ACGGTGTTCTGTTTCTCC 77 47                                   - 19503 Coding 786 CTACAGGAGACACACTCG 81 48                                   - 19504 Coding 796 CTTACAGTTACTACAGGA 61 49                                   - 19505 Coding 802 GCTTTTCTTACAGTTACT 93 50                                   - 19506 Coding 807 TCCAGGCTTTTCTTACAG 71 51                                   - 19507 Coding 845 TAACATTCTCAATCTGGG 0 52                                    - 19508 Coding 873 ACTGTGGTGCCTGAGTCC 74 53                                   - 19509 Coding 906 CAAAGACCAAAGAAAATG 29 54                                   - 19510 Coding 911 AAAGGCAAAGACCAAAGA 45 55                                   - 19511 Coding 921 AGGAGGGATAAAAGGCAA 67 56                                   - 19512 Coding 929 CAATGAAGAGGAGGGATA 55 57                                   - 19513 Coding 935 TTAAACCAATGAAGAGGA 25 58                                   - 19514 Coding 952 CCGTTGGTAGCGATACAT 93 59                                   - 19515 Coding 992 TCGATTTCCCACAAACAA 16 60                                   - 19516 Coding 1033 CTTAGTAGTAGTTCCTTC 70 61                                  - 19517 Coding 1075 GAAGCCTGGAGTGGGACT 0 62                                   - 19518 Coding 1098 GGACTGAAGCCCAGGGTG 31 63                                  - 19519 Coding 1113 GTGGAACTGGGCACGGGA 41 64                                  - 19520 Coding 1118 TGAAGGTGGAACTGGGCA 51 65                                  - 19521 Coding 1127 AGCTGGAGGTGAAGGTGG 59 66                                  - 19522 Coding 1162 CGCAAAGTTGGGACAGTC 80 67                                  - 19523 Coding 1184 GTGCCACCTCTCTGCGGG 40 68                                  - 19524 Coding 1269 CTGTCCTCCCACTTCTGA 67 69                                  - 19525 Coding 1290 AGGCTCTGTGGCTTGTGG 79 70                                  - 19526 Coding 1389 TCGTGGTCGCTCAGCCCT 75 71                                  - 19527 Coding 1465 CCGCCTCCAGGTCGCCAG 57 72                                  - 19528 Coding 1537 GCAGCCCAGCAGGTCCAT 68 73                                  - 19529 Coding 1545 TCCTCCAGGCAGCCCAGC 80 74                                  - 19530 Coding 1604 ATCTGAGAAGACTGGGCG 19 75                                  - 19531 Coding 1707 GCTCCTGCTTGCCCCTGC 89 76                                  - 19532 Coding 1732 GTTAGCACCAAGTAGGCG 80 77                                  - 19533 Coding 1842 CGCAAACCACCCACTCAG 79 78                                  - 19534 Coding 1847 ATCCTCGCAAACCACCCA 42 79                                  - 19535 Coding 1859 ATAGCGTCCCTCATCCTC 52 80                                  - 19536 Coding 1925 CTCAGGGACGAACCAGGG 92 81                                  - 19537 Coding 1930 AAAGGCTCAGGGACGAAC 41 82                                  - 19538 Coding 1979 ACAAAACAAAACAAAACA 0 83                                   - 19539 Coding 2016 GCCAAGTTTCTATTAGTG 87 84                                  - 19540 Coding 2033 GCAGAGGGCACAGGAGTG 59 85                                  - 19541 Coding 2039 GTCCAGGCAGAGGGCACA 72 86                                  - 19542 Coding 2043 GCTTGTCCAGGCAGAGGG 58 87                                  - 19543 Coding 2071 TGCCTTAGGACAGTTCAG 69 88                                  - 19544 Coding 2085 TCCGTGCTCGCCCCTGCC 62 89                                  - 19545 Coding 2089 TTGTTCCGTGCTCGCCCC 57 90                                  - 19546 Coding 2097 AGGCCCCATTGTTCCGTG 79 91                               ______________________________________                                    

As shown in Table 2, SEQ ID NOs 11, 15 16, 17, 21, 22, 23, 28, 29, 30,31, 33, 35, 36, 37, 38, 39, 41, 43, 46, 47, 48, 50, 59, 67, 70, 71, 74,76, 77, 78, 81, 84 and 91 demonstrated at least 75% inhibition of TNFR1expression in this experiment and are therefore preferred.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 91                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2161                                                              (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - CGGCCCAGTG ATCTTGAACC CCAAAGGCCA GAACTGGAGC CTCAGTCCAG  - #                  50                                                                        - - AGAATTCTGA GAAAATTAAA GCAGAGAGGA GGGGAGAGAT CACTGGGACC  - #                 100                                                                         - - AGGCCGTGAT CTCTATGCCC GAGTCTCAAC CCTCAACTGT CACCCCAAGG  - #                 150                                                                         - - CACTTGGGAC GTCCTGGACA GACCGAGTCC CGGGAAGCCC CAGCACTGCC  - #                 200                                                                         - - GCTGCCACAC TGCCCTGAGC CCAAATGGGG GAGTGAGAGG CCATAGCTGT  - #                 250                                                                         - - CTGGCATGGG CCTCTCCACC GTGCCTGACC TGCTGCTGCC GCTGGTGCTC  - #                 300                                                                         - - CTGGAGCTGT TGGTGGGAAT ATACCCCTCA GGGGTTATTG GACTGGTCCC  - #                 350                                                                         - - TCACCTAGGG GACAGGGAGA AGAGAGATAG TGTGTGTCCC CAAGGAAAAT  - #                 400                                                                         - - ATATCCACCC TCAAAATAAT TCGATTTGCT GTACCAAGTG CCACAAAGGA  - #                 450                                                                         - - ACCTACTTGT ACAATGACTG TCCAGGCCCG GGGCAGGATA CGGACTGCAG  - #                 500                                                                         - - GGAGTGTGAG AGCGGCTCCT TCACCGCTTC AGAAAACCAC CTCAGACACT  - #                 550                                                                         - - GCCTCAGCTG CTCCAAATGC CGAAAGGAAA TGGGTCAGGT GGAGATCTCT  - #                 600                                                                         - - TCTTGCACAG TGGACCGGGA CACCGTGTGT GGCTGCAGGA AGAACCAGTA  - #                 650                                                                         - - CCGGCATTAT TGGAGTGAAA ACCTTTTCCA GTGCTTCAAT TGCAGCCTCT  - #                 700                                                                         - - GCCTCAATGG GACCGTGCAC CTCTCCTGCC AGGAGAAACA GAACACCGTG  - #                 750                                                                         - - TGCACCTGCC ATGCAGGTTT CTTTCTAAGA GAAAACGAGT GTGTCTCCTG  - #                 800                                                                         - - TAGTAACTGT AAGAAAAGCC TGGAGTGCAC GAAGTTGTGC CTACCCCAGA  - #                 850                                                                         - - TTGAGAATGT TAAGGGCACT GAGGACTCAG GCACCACAGT GCTGTTGCCC  - #                 900                                                                         - - CTGGTCATTT TCTTTGGTCT TTGCCTTTTA TCCCTCCTCT TCATTGGTTT  - #                 950                                                                         - - AATGTATCGC TACCAACGGT GGAAGTCCAA GCTCTACTCC ATTGTTTGTG  - #                1000                                                                         - - GGAAATCGAC ACCTGAAAAA GAGGGGGAGC TTGAAGGAAC TACTACTAAG  - #                1050                                                                         - - CCCCTGGCCC CAAACCCAAG CTTCAGTCCC ACTCCAGGCT TCACCCCCAC  - #                1100                                                                         - - CCTGGGCTTC AGTCCCGTGC CCAGTTCCAC CTTCACCTCC AGCTCCACCT  - #                1150                                                                         - - ATACCCCCGG TGACTGTCCC AACTTTGCGG CTCCCCGCAG AGAGGTGGCA  - #                1200                                                                         - - CCACCCTATC AGGGGGCTGA CCCCATCCTT GCGACAGCCC TCGCCTCCGA  - #                1250                                                                         - - CCCCATCCCC AACCCCCTTC AGAAGTGGGA GGACAGCGCC CACAAGCCAC  - #                1300                                                                         - - AGAGCCTAGA CACTGATGAC CCCGCGACGC TGTACGCCGT GGTGGAGAAC  - #                1350                                                                         - - GTGCCCCCGT TGCGCTGGAA GGAATTCGTG CGGCGCCTAG GGCTGAGCGA  - #                1400                                                                         - - CCACGAGATC GATCGGCTGG AGCTGCAGAA CGGGCGCTGC CTGCGCGAGG  - #                1450                                                                         - - CGCAATACAG CATGCTGGCG ACCTGGAGGC GGCGCACGCC GCGGCGCGAG  - #                1500                                                                         - - GCCACGCTGG AGCTGCTGGG ACGCGTGCTC CGCGACATGG ACCTGCTGGG  - #                1550                                                                         - - CTGCCTGGAG GACATCGAGG AGGCGCTTTG CGGCCCCGCC GCCCTCCCGC  - #                1600                                                                         - - CCGCGCCCAG TCTTCTCAGA TGAGGCTGCG CCCCTGCGGG CAGCTCTAAG  - #                1650                                                                         - - GACCGTCCTG CGAGATCGCC TTCCAACCCC ACTTTTTTCT GGAAAGGAGG  - #                1700                                                                         - - GGTCCTGCAG GGGCAAGCAG GAGCTAGCAG CCGCCTACTT GGTGCTAACC  - #                1750                                                                         - - CCTCGATGTA CATAGCTTTT CTCAGCTGCC TGCGCGCCGC CGACAGTCAG  - #                1800                                                                         - - CGCTGTGCGC GCGGAGAGAG GTGCGCCGTG GGCTCAAGAG CCTGAGTGGG  - #                1850                                                                         - - TGGTTTGCGA GGATGAGGGA CGCTATGCCT CATGCCCGTT TTGGGTGTCC  - #                1900                                                                         - - TCACCAGCAA GGCTGCTCGG GGGCCCCTGG TTCGTCCCTG AGCCTTTTTC  - #                1950                                                                         - - ACAGTGCATA AGCAGTTTTT TTTGTTTTTG TTTTGTTTTG TTTTGTTTTT  - #                2000                                                                         - - AAATCAATCA TGTTACACTA ATAGAAACTT GGCACTCCTG TGCCCTCTGC  - #                2050                                                                         - - CTGGACAAGC ACATAGCAAG CTGAACTGTC CTAAGGCAGG GGCGAGCACG  - #                2100                                                                         - - GAACAATGGG GCCTTCAGCT GGAGCTGTGG ACTTTTGTAC ATACACTAAA  - #                2150                                                                         - - ATTCTGAAGT T               - #                  - #                      - #     2161                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - GCTTCAGAAA ACCACCTCAG ACA           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - CCGGTCCACT GTGCAAGAA             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - TCAGCTGCTC CAAATGCCGA AAGG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - GAAGGTGAAG GTCGGAGTC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - GAAGATGGTG ATGGGATTTC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - CAAGCTTCCC GTTCTCAGCC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - TTCTCTGGAC TGAGGCTC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - TCCCCTCCTC TCTGCTTT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - AGACTCGGGC ATAGAGAT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - GGTTGAGACT CGGGCATA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                          - - TGAGGGTTGA GACTCGGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                          - - ACAGTTGAGG GTTGAGAC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                          - - GGTGACAGTT GAGGGTTG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15:                          - - GCAGTGTGGC AGCGGCAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #16:                          - - AGGGCAGTGT GGCAGCGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #17:                          - - CTCAGGGCAG TGTGGCAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #18:                          - - TTGGGCTCAG GGCAGTGT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #19:                          - - CATTTGGGCT CAGGGCAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #20:                          - - GTCAGGCACG GTGGAGAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #21:                          - - GCAGGTCAGG CACGGTGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #22:                          - - GCAGCAGCAG GTCAGGCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #23:                          - - AGCGGCAGCA GCAGGTCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #24:                          - - CACCAGCGGC AGCAGCAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #25:                          - - CAGGAGCACC AGCGGCAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #26:                          - - TATATTCCCA CCAACAGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #27:                          - - TCTTCTCCCT GTCCCCTA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #28:                          - - ATTATTTTGA GGGTGGAT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #29:                          - - GTTCCTTTGT GGCACTTG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #30:                          - - AGTAGGTTCC TTTGTGGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #31:                          - - GCCTGGACAG TCATTGTA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #32:                          - - CAGTCCGTAT CCTGCCCC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #33:                          - - AGCCGCTCTC ACACTCCC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #34:                          - - TCTGAAGCGG TGAAGGAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #35:                          - - GGTTTTCTGA AGCGGTGA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #36:                          - - AGGTGGTTTT CTGAAGCG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #37:                          - - GTCTGAGGTG GTTTTCTG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #38:                          - - AGGCAGTGTC TGAGGTGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #39:                          - - AGCTGAGGCA GTGTCTGA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #40:                          - - CATTTCCTTT CGGCATTT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #41:                          - - GACCCATTTC CTTTCGGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 42:                        - - CACCTGACCC ATTTCCTT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #43:                          - - GGTACTGGTT CTTCCTGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #44:                          - - TTTTCACTCC AATAATGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #45:                          - - CCATTGAGGC AGAGGCTG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #46:                          - - ACGGTCCCAT TGAGGCAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #47:                          - - ACGGTGTTCT GTTTCTCC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #48:                          - - CTACAGGAGA CACACTCG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #49:                          - - CTTACAGTTA CTACAGGA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #50:                          - - GCTTTTCTTA CAGTTACT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #51:                          - - TCCAGGCTTT TCTTACAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #52:                          - - TAACATTCTC AATCTGGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #53:                          - - ACTGTGGTGC CTGAGTCC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #54:                          - - CAAAGACCAA AGAAAATG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #55:                          - - AAAGGCAAAG ACCAAAGA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #56:                          - - AGGAGGGATA AAAGGCAA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #57:                          - - CAATGAAGAG GAGGGATA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #58:                          - - TTAAACCAAT GAAGAGGA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #59:                          - - CCGTTGGTAG CGATACAT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #60:                          - - TCGATTTCCC ACAAACAA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #61:                          - - CTTAGTAGTA GTTCCTTC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #62:                          - - GAAGCCTGGA GTGGGACT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #63:                          - - GGACTGAAGC CCAGGGTG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #64:                          - - GTGGAACTGG GCACGGGA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #65:                          - - TGAAGGTGGA ACTGGGCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #66:                          - - AGCTGGAGGT GAAGGTGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #67:                          - - CGCAAAGTTG GGACAGTC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #68:                          - - GTGCCACCTC TCTGCGGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #69:                          - - CTGTCCTCCC ACTTCTGA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #70:                          - - AGGCTCTGTG GCTTGTGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:71:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #71:                          - - TCGTGGTCGC TCAGCCCT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:72:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #72:                          - - CCGCCTCCAG GTCGCCAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:73:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #73:                          - - GCAGCCCAGC AGGTCCAT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:74:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #74:                          - - TCCTCCAGGC AGCCCAGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:75:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #75:                          - - ATCTGAGAAG ACTGGGCG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:76:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #76:                          - - GCTCCTGCTT GCCCCTGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:77:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #77:                          - - GTTAGCACCA AGTAGGCG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:78:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #78:                          - - CGCAAACCAC CCACTCAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:79:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #79:                          - - ATCCTCGCAA ACCACCCA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:80:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #80:                          - - ATAGCGTCCC TCATCCTC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:81:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #81:                          - - CTCAGGGACG AACCAGGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:82:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #82:                          - - AAAGGCTCAG GGACGAAC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:83:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #83:                          - - ACAAAACAAA ACAAAACA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:84:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #84:                          - - GCCAAGTTTC TATTAGTG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:85:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #85:                          - - GCAGAGGGCA CAGGAGTG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:86:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #86:                          - - GTCCAGGCAG AGGGCACA             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:87:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #87:                          - - GCTTGTCCAG GCAGAGGG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:88:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #88:                          - - TGCCTTAGGA CAGTTCAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:89:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #89:                          - - TCCGTGCTCG CCCCTGCC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:90:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #90:                          - - TTGTTCCGTG CTCGCCCC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:91:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #91:                          - - AGGCCCCATT GTTCCGTG             - #                  - #                      - #  18                                                                 __________________________________________________________________________

What is claimed is:
 1. An antisense compound up to 30 nucleobases inlength comprising SEQ ID NO: 11, 15, 16, 17, 21, 22, 23, 25, 28, 29, 30,31, 33, 35, 36, 37, 38, 39, 41, 42, 43, 45, 46, 47, 48, 50, 59, 62, 67,70, 71, 74, 76, 77, 78, 81, 82, 84, 90 or
 91. 2. The antisense compoundof claim 1 which is an antisense oligonucleotide.
 3. The antisensecompound of claim 2 which comprises at least one modifiedinternucleoside linkage.
 4. The antisense compound of claim 3 whereinthe modified internucleoside linkage is a phosphorothioate linkage. 5.The antisense compound of claim 2 which comprises at least one modifiedsugar moiety.
 6. The antisense compound of claim 5 wherein the modifiedsugar moiety is a 2'-O-methoxyethyl sugar moiety.
 7. The antisensecompound of claim 2 which comprises at least one modified nucleobase. 8.The antisense compound of claim 7 wherein the modified nucleobase is a5-methylcytosine.
 9. The antisense compound of claim of claim 2 which isa chimeric oligonucleotide.
 10. A composition comprising the antisensecompound of claim 1 and a carrier or diluent.
 11. A method of inhibitingthe expression of the TNFR1 in human cells or tissues comprisingcontacting said human cells or tissues with the antisense compound ofclaim 1 in vitro so that expression of human TNFR1 is inhibited.